icacgm18th International Colloquim of Animal Cytogenetics and Gene Mapping 2008

Session I - Chromosome Architecture

Lectures (L), Oral communications (O), Posters (P)


Chromosomal and nuclear genome architecture - an evolutionary perspective

Stefan Mueller

Department Biology II, Human Genetics, Ludwig-Maximilians University, Munich (Germany)

To date, over 100 mammalian species were investigated by cross-species chromosome painting. Large-insert cloned probes have been used for the establishment of high-resolution comparative FISH maps and for a detailed analysis of evolutionary breakpoints. The resulting chromosomal homology maps provided insight into the evolutionary history of each human chromosome. This knowledge has been prerequisite to address open questions concerning evolutionary conserved and species-specific principles of the three-dimensional chromosome organisation in the interphase nucleus and its possible consequences on the direction of chromosome and genome evolution.

This presentation gives a brief reconstruction of the major landmarks in primate karyotype evolution, including some recent results obtained from the analysis of evolutionary breakpoints. I will then summarize the present knowledge on the interplay between the linear organization of the mammalian genome on the metaphase chromosome and the non-random spatial arrangement of entire chromosomes and of individual loci in the interphase nucleus. The impact of evolutionary translocations, fusions, fissions and inversions on the 3D-conformation of chromosome territories and on the positioning of breakpoint flanking loci will be discussed. Finally I will provide examples where non-random nuclear neighborhoods in male meiosis potentially could have triggered evolutionary rearrangements.


Satellite DNA: Hallmarks of Chromosome Evolution and Genome Remodelling

Raquel Chaves

Institute for Biotechnology and Bioengineering, Centre of Genetics and Biotechnology of the University of Trás-os-Montes and Alto Douro (IBB/CGB-UTAD), 5001-801 Vila Real, (Portugal).

Heterochromatic regions harbour satellite DNA sequences which are a very dynamic component of mammalian genomes, constituting an important factor of genomic plasticity. Recent research provide a large growing body of evidence indicating that tandem repetitive sequences and satellite DNA play an important role in mammalian evolution by promoting chromosomal rearrangements. Moreover, satellite DNAs and their transcripts seem to have an active regulatory role in eukaryotic organisms; chromatin modulation and control of gene expression are some of the traits in which satellite DNAs could be involved.

Different satellite sequences co-exist in the genome, forming a satellite DNA library made of independent evolutionary units that are ruled by the mechanisms of concerted evolution, leading to the emergence of species-specific satellite profiles. Changes in satellite DNA can be correlated with chromosomal evolution and influence the evolution of species.

There are several conjectures portrayed to explain the active role of satellite DNA in genomes remodeling. Since the breakpoints occur in the repetitive DNA blocks, the chromosome rearrangements would have low effects on the euchromatic genome by keeping syntenic segments intact. Other intriguing fact is that in the different mammalian evolutionary pathways subsequent incorporation of satellite DNA sequences occurs at repositioned centromere sites, but what is the evolutionary advantage of having repetitive DNA at centromeres remains a mystery. Some of the suggestions for this evidence are that the satellite DNA at centromeres might increase the loading of constitutive proteins, or promote a heterochromatic environment more favourable for sister chromatid cohesion. However, further experiments are needed to depict conclusive evidences about the causal involvement of satellite DNA in chromosomal evolution and will certainly allow the ascertainment of the mechanisms that effectively explain the role of satellite DNA in chromosome and in genome evolution.
Here, special emphasis will be given to the "hallmarks" that constitute true evidences of the involvement of heterochromatic regions and satellite DNA in the evolution of chromosomes and in genomes’ remodelling. The value of satellite DNA markers in the reconstruction of group phylogenies, models for chromosome rearrangement and implications in function will be highlighted with examples from various mammalian groups such as Cetartiodactyla, Rodentia and Carnivora.


Changes in gene position in interphase during porcine adipogenesis are correlated with changes in gene expression

I. Szczerbal (1), H.A. Foster (2), J.M. Bridger (2)

(1) Department of Genetics and Animal Breeding, Agricultural University of Poznan, Poznan (Poland)
(2) Centre for Cell and Chromosome Biology, Division of Biosciences, Brunel University, London (UK)

Differentiation is characterized by dynamic changes in nuclear morphology as well as dramatic changes in the gene expression profile. We wished to determine if changes in gene expression are correlated with alterations to the organization of the genome in interphase nuclei. In this study we investigated the nuclear position of selected genes involved in adipogenesis and the chromosomes they are housed on during adipogenesis in the pig. Knowledge about cellular aspects of the formation of fat cells is very important since the pig plays a central role in agricultural sciences and is also considered as a model for human obesity. We have established a system for differentiation of porcine mesenchymal stem cells into adipocytes and have analysed the spatial position of nine genes. Seven of these genes are associated with adipogenesis (PPARG, CEBPA, CEBPB, SREBP, CREB, GATA2, FABP4) and two genes were used as a control (SOX9, MYL1). Additionally, positions of chromosomes carrying the studied gene loci (SSC4, SSC6, SSC12, SSC13, SSC15 and SSC17) were determined. It has been found that during adipogenesis in our system the nuclear position of genes is altered, with upregulation of gene expression correlating with genes becoming more internally located. However, the positions of chromosome territories remained unchanged. It seems that the temporal repositioning of the genes during adipogenesis is correlated with gene activity.

(This study was financed by the Polish Ministry of Education and Science, grant N301 3381 33 and 9/MOB/2007/0).


Application of in situ fluorescent nick - translation to detection DNA methylation in B chromosomes of red fox (Vulpes vulpes) and Chinese raccoon dog (Nyctereutes procyonoides procyonoides)

Ewa Słota (1), Maciej Wnuk (2), Monika Bugno (1), Aldona Pieńkowska - Schelling (3)

(1) Department of Immuno- and Cytogenetics, National Research Institute of Animal Production, Balice/Kraków (Poland)
(2) Department of Genetics, University of Rzeszów, Rzeszów (Poland)
(3) Swiss Federal Institute of Technology Zurich, Departament of Animal Science (Switzerland)

B chromosomes are additional, chromosomes to the standard complement that occur in natural populations of many different species. Red fox (Vulpes vulpes; 2n = 34 + 0-7 B) and Chinese raccoon dog (Nyctereutes procyonoides procyonoides; 2n=54 + 0-4 B) are two mammalian species with B chromosomes. Although molecular analyses have revealed that the B chromosomes from both species may be composed of various repetitive DNA sequences such as rDNA or (TTAGGG)n, many questions regarding their origin and function still remain open. In this study we have applied in situ fluorescent nick - translation method to investigate the distribution of CpG sequences along B chromosomes. This method is based on a digestion of nonmethylated CpG by restriction endonuclease Hpa II followed by a detection of chromosomal location of these sequences in the presence of Taq polymerase and labeled nucleotides. We found DNA methylation in a both B chromosomes of Chinese raccoon dog. However B chromosomes of red fox appeared to have different DNA methylation pattern. One of B chromosomes of red fox was weakly labeled whereas the other was not labeled at all. In our opinion this method gives satisfactory results and it is attractive complement to other cytogenetics techniques applied to investigate the structure of B chromosomes.


Porcine and bovine centromeric and pericentromeric sequences

H. Hayes (1), M. Stam (2), M. Bertaud (1), A. Eggen (1), M. Yerle (3), C. Rogel-Gaillard (2)

(1) UR339 INRA, 78350 Jouy-en-Josas (France) ;
(2) UMR314, INRA CEA, 78350 Jouy-en-Josas (France) ;
(3) UMR444, INRA ENVT, 31326 Castanet-Tolosan (France)

In eukaryotes, centromere regions are essential for accurate chromosome segregation during mitosis and meiosis and assemble at a unique DNA locus on each chromosome. The sequences of the pig and bovine genomes are not yet complete, and major gaps remain at the centromeric and pericentromeric regions of each chromosome, which correspond to repetitive satellite DNA that is either not efficiently subcloned or not assembled via whole-genome assembly techniques. Satellite DNA is composed of heterogeneous repeated DNA motifs characterized by their sequence, size and number, which differ among species and to a lesser extent among chromosomes. In man, based on the polymorphism of alpha satellite DNA, chromosome-specific centromeric probes are commercialised, which are highly useful for diagnosis purposes because of their intense FISH signals. We have started a project to develop such centromeric probes in pig and bovine. Towards this aim, 13 pig and two bovine BAC clones hybridising specifically to the centromeric/pericentromeric regions of all or part of the pig and bovine chromosomes have been sequenced (3X coverage). After assembling the sequences with the PHRED and PHRAP programs, contigs were visualised with CONSED and known DNA repeats detected by RepeatMasker. The results show that in both species, these BAC contain different families of repeat motifs and constitute a reservoir of potential centromeric and pericentromeric probes.


A detailed Constitutive Heterochromatin Map for Praomys tullbergi (Rodentia, Muridae) karyotype

S. Meles, A. Paco, F. Adega, H. Guedes-Pinto, R. Chaves

Institute for Biotechnology and Bioengineering, Centre of Genetics and Biotechnology, UTAD (IBB/CGB-UTAD), 1013, 5001-801 Vila Real (Portugal)

Constitutive heterochromatin (CH) is usually identified by classical C-banding. However, for a more detailed CH characterization, in terms of its location, detection of different subclasses and molecular composition, other methodologies are necessary. Among these, base-specific fluorochromes technique, which delimit chromosome regions rich in different types of DNA bases and in situ Restriction Endonuclease (REs) digestion, which cleave DNA at specific sequences, followed by C-banding, proved to be very useful.
In the present work, the CH of Praomys tullbergi (PTU) was detailed described, using specific fluorochromes for DNA regions rich in AT (DAPI) or GC (CMA3) and a panel of seven REs, followed by C-banding. These combined methodologies allowed the detection of 52 CH subclasses, distributed by three classes: centromeric, interstitial and telomeric.

It is now accepted that CH regions are hotspots for structural rearrangements being implicated in chromosome evolution. The CH map constructed can be used to predict the chromosomal rearrangements involved in the evolution of this genome. Constitutive Heterochromatin Maps can thus be used to analyse the chromosomal architecture of genomes.

This work was supported by a project POCI/BIA-BMC/58541/2004, and two PhD and one Pos-doc grants, SFRH/BD/41576/2007, SFRH/BD/41574/2007 and SFRH/BPD/32661/2006 of the Science and Technology Foundation of Portugal. We thank Dr. V. Volobouev for the Rodentia cell cultures.


Comparative analysis of structural organisation of mouse repeated sequences

A. Fedorov, O. Podgornaya

Institute of cytology RAS, Tikhoretsky pr. 4, Saint-Petersburg (Russia)

Investigation of genomes of higher eukaryotes showed that they contain considerable amount of repetitive DNA sequences. Role of these DNA repeats in the genome functioning still remains illusive. Repetitive DNA sequences make up about 50% of mammalian genomes, whereas genes - less than 5%. Functionally important classes of DNA repeats include tandemly repeated sequences, which are involved into the organisation of centromeric regions of chromosomes, and interspersed repeats, such as LINE1, SINE and LTR retrotransposons, which are involved into genome rearrangements and influence gene expression.

Detection and complex analysis of structural organisation and distribution of LINE1, LTR and SINE retrotransposons in the assembled contigs of mouse genome, comparatively with the distribution of known heterochromatin regions and centromeric satellite tandem repeats, were performed in the present work. The results of computer analysis were verified with the help of PCR, Southern hybridisation and FISH on the metaphase and interphase chromosomes. Absence of interspersed repeats in chromocenters indicates that these nuclear domains are represented by chromosome regions formed by satellite DNA only. Genomic LINE1s are characterized by 5’-truncations and inverted structures that could result in generation of double-stranded RNA. In contrast to LTR retrotransposons most genomic copies of LINE1 repeats are truncated at their 5’-ends and thus do not contain promoters. In the interphase nucleus full length promoter containing LINE1s preferentially localized in several foci.